Activity of recombinant tumor necrosis factor on Toxoplasma gondii and Trypanosoma cruzi

Activated macrophages produce tumor necrosis factor (TNF), a cytokine with anti-tumor and anti-plasmodia activities. This study revealed that recombinant TNF (rTNF) inhibits intracellular multiplication of blood trypomastigotes of Trypanosoma cruzi in murine peritoneal macrophages. rTNF did not have any apparent direct effect on the survival of extracellular T. cruzi or on its ability to infect mammalian cells. The degree of inhibition of the intracellular multiplication of T. cruzi was found to be a function of the time of exposure of the infected cells to rTNF. rTNF induced a comparable effect when different strains of the parasite were used. In contrast to its activity on T. cruzi, rTNF did not affect intracellular multiplication of Toxoplasma gondii tachyzoites or bradyzoites in normal murine peritoneal macrophages or in human fibroblasts. Killing of Toxoplasma tachyzoites by activated macrophages was not enhanced by rTNF.     

Trypanosoma cruzi: isolation of cloned strains and characterization of their infectivity

Cloning of Trypanosoma cruzi strains Y, CL and Colombiana was achieved by plating on solid medium. Clones were obtained either from culture epimastigotes or from bloodstream trypomastigotes. In both cases the efficiency of plating was almost 100%. Clones from culture epimastigotes did not infect the albino mouse, while clones from bloodstream trypomastigotes remained infective even after several passages in a blood-agar/BHI biphasic medium, in which the amastigote-like forms prevail.     

Sialoglycoproteins and sialoglycolipids contribute to the negative surface charge of epimastigote and trypomastigote forms of Trypanosoma cruzi

Epimastigote and trypomastigote forms of Trypanosoma cruzi have a net negative surface charge, as determined by direct measurement of the mean cellular electrophoretic mobility. Treatment of the parasites with neuraminidase reduces by 17 and 52% the mean electrophoretic mobility of epimastigote and bloodstream trypomastigote forms, respectively. Neuraminidase-treated cells recover their normal electrophoretic mobility if incubated for 2 h in the presence of fresh culture medium. The recovering process of epimastigotes is almost totally blocked by addition of inhibitors of either protein synthesis (puromycin) or N-glycosidically linked glycoprotein synthesis (tunicamycin). The recovering process of trypomastigotes is not totally inhibited by either puromycin or tunicamycin. Treatment of T. cruzi with trypsin reduces by 11 and 40% the mean electrophoretic mobility of epimastigote and bloodstream trypomastigote forms. Trypsin-treated cells recover their normal electrophoretic mobility if incubated for 4 h in fresh culture medium. The recovering process of trypomastigotes is partially inhibited by puromycin. The results obtained indicate that sialoglycoproteins and sialoglycolipids exist on the surface of T. cruzi, the latter being predominant on the surface of trypomastigotes.     

Trypanosoma cruzi: T-cell-dependent mechanisms of resistance during chronic infection

Effector mechanisms of resistance exerted by T cells from BALB/c mice chronically infected with Trypanosoma cruzi, Tulahuén strain, were studied. Spleen cells from chronically infected mice (Chro-SC) prestimulated with heat-killed trypomastigotes (HKT) and/or IL-2 destroyed PHA-labeled p-815 mastocytoma cells, HKT-pulsed macrophages, and normal peritoneal macrophages. However, HKT-stimulated Chro-SC did not affect the infectivity of free bloodstream forms of the parasite. Upon HKT stimulation, Chro-SC or their culture supernatant activated peritoneal macrophages for the destruction of intracellular amastigotes. The effect was abolished after Thy 1.2+ cell depletion. The addition of Cyclosporin A (CyA), which blocks T-cell activation, during HKT-stimulation of Chro-SC, diminished their ability to activate the trypanocidal activity of macrophages. CyA also inhibited the production of both macrophage-activating factors and interferon-gamma by HKT-stimulated Chro-SC. CyA administration to recipients of nylon-wool nonadherent spleen cells from chronically infected mice inhibited their adoptively acquired resistance against T. cruzi, suggesting that the conferred resistance depended on the effect of specifically activated cells. When administered during the chronic stage of the infection, CyA abrogated the antigen-specific delayed type hypersensitivity response but increased the levels of anti-T. cruzi IgG antibodies. Neither parasitemia, tissular parasitism in myocardium or skeletal muscle, nor mortality were detected after CyA treatment, suggesting the presence of a CyA nonsensitive mechanism(s) in the control of T. cruzi during the chronic phase of the infection.     

Trypanosoma cruzi: differences in cell surface interaction of circulating (trypomastigote) and culture (epimastigote) forms with macrophages

Characteristics of the association of circulating (trypomastigote) and cultured (epimastigote) forms of Trypanosoma cruzi with macrophages were studied. Treatment of mouse macrophages with the anti-microfilament drug cytochalasin D severely reduced the ability of these cells to bind either trypomastigotes or epimastigotes. Instead, treatment with the antimicrotubule drug colchicine or 2-deoxyglucose afforded differential effects because epimastigote but not trypomastigote association with the macrophages was significantly inhibited. Prior treatment of epimastigotes with either trypsin or neuraminidase decreased their uptake by macrophages whereas treatment of trypomastigotes with either enzyme increased it. Pretreatment of macrophages with neuraminidase did not affect epimastigote uptake but reduced that of trypomastigotes. Pretreatment of macrophages with trypsin reduced the uptake of both forms of the parasite. However, quantitative differences in the extent of such reduction were noted when varying concentrations of trypsin were used, epimastigote uptake being more drastically affected. These results suggest that the initial interaction of virulent circulating trypomastigote and the attenuated cultured epimastigote forms of T. cruzi to macrophages may involve attachment via different surface structures.     

The interaction of myotropic and macrophagotropic strains of Trypanosoma cruzi with myoblasts and fibers of skeletal muscle

The process of interaction of bloodstream trypomastigotes from the myotropic CL and Colombiana strains and the macrophagotropic Y strain of Trypanosoma cruzi with mouse myoblasts and myotubes was analysed. After 24 h of parasite-host cell interaction, parasites from the CL and Colombiana strains appeared to be more infective to myoblasts than those from the Y strain. Parasites from the Colombiana strain were more infective for myotubes than those from the Y strain, while those from the CL strain showed very a low ability to infect the cells. For all strains the infectivity was low for short periods of interaction, increasing with time. Myoblasts infected with parasites from the Y strain fused with other infected and uninfected cells to form myotubes. However, the process of fusion was blocked when the myoblasts were infected with parasites from the CL and Colombiana strains. These data indicate a different behavior of muscle cells when in contact with myotropic or non-myotropic strains of T. cruzi.     

Rab5 activation by Toll-like receptor 2 is required for Trypanosoma cruzi internalization and replication in macrophages

Trypanosoma cruzi can infect and replicate in macrophages. During invasion, T. cruzi interacts with different macrophage receptors to induce its own phagocytosis. However, the nature of those receptors and the molecular mechanisms involved are poorly understood. In this study, we demonstrate that T. cruzi metacyclic trypomastigotes but not epimastigotes were able to induce Rab5 activation and binding to the early endosomes in peritoneal macrophages. In this process, active Rab5 colocalized with parasites in the phagosome and with the Rab5A effector molecule early endosomal antigen 1. Phagosome formation and T. cruzi internalization were inhibited in Raw 264.7 macrophages expressing a dominant-negative form of Rab5 [(S34N)Rab5]. Using T. cruzi membrane extracts, we verified that the Rab5 activation depends on the interaction between parasite surface molecules and macrophages surface molecule. In addition, during infection of macrophages, phosphatidylinositol 3-kinase (PI3K) pathway was activated. Assays carried out using a selective PI3K inhibitor (LY294002) showed that the PI3K activation is essential for Rab5 activation by T. cruzi infection and for the entrance and intracellular replication of T. cruzi in macrophages. Moreover, using macrophages from knockout mice, we found that activation of Rab5, fusion of early endosomes and phagocytosis induced by T. cruzi infection involved Toll-like receptor (TLR)2 but were independent of TLR4 receptors.     

Primaquine is lethal for intracellular but not extracellular Trypanosoma cruzi

Primaquine has been used to treat Chagas' disease in humans and has been reported to be active against extracellular Trypanosoma cruzi. Experiments were designed to evaluate the relative activity of primaquine against extra- and intracellular T. cruzi and to determine if primaquine might be combined advantageously with ketoconazole. Primaquine at 0.5 micrograms/ml significantly inhibited T. cruzi replication in infected mouse peritoneal macrophages and also effectively treated infected L929 cells. To examine the effect of primaquine on extracellular organisms, tissue culture T. cruzi were incubated with primaquine for different periods of time and then used to infect macrophages. Incubation with 10 micrograms/ml for 14 hr but not 8 hr significantly inhibited but did not eradicate replication. Incubation of spleen amastigotes or blood trypomastigotes for 2 hr with 10 micrograms/ml did not inhibit replication. Incubation of extracellular tissue culture T. cruzi with primaquine for 2 hr did not potentiate the activity of ketoconazole against intracellular organisms. The combination of primaquine and ketoconazole administered to acutely infected mice significantly decreased parasitemias in comparison to treatment with primaquine or ketoconazole alone. Thus primaquine acts primarily on intracellular rather than extracellular T. cruzi. Primaquine and ketoconazole appear to have additive activity in vivo.     

Molecular and cellular mechanisms involved in the Trypanosoma cruzi/host cell interplay

The protozoan parasite Trypanosoma cruzi has a complex biological cycle that involves vertebrate and invertebrate hosts. In mammals, the infective trypomastigote form of this parasite can invade several cell types by exploiting phagocytic-like or nonphagocytic mechanisms depending on the class of cell involved. Morphological studies showed that when trypomastigotes contact macrophages, they induce the formation of plasma membrane protrusions that differ from the canonical phagocytosis that occurs in the case of noninfective epimastigotes. In contrast, when trypomastigotes infect epithelial or muscle cells, the cell surface is minimally modified, suggesting the induction of a different class of process. Lysosomal-dependent or -independent T. cruzi invasion of host cells are two different models that describe the molecular and cellular events activated during parasite entry into nonphagocytic cells. In this context, we have previously shown that induction of autophagy in host cells before infection favors T. cruzi invasion. Furthermore, we demonstrate that autophagosomes and the autophagosomal protein LC3 are recruited to the T. cruzi entry sites and that the newly formed T. cruzi parasitophorous vacuole has characteristics of an autophagolysosome. This review summarizes the current knowledge of the molecular and cellular mechanisms of T. cruzi invasion in nonphagocytic cells. Based on our findings, we propose a new model in which T. cruzi takes advantage of the upregulation of autophagy during starvation to increase its successful colonization of host cells.     

Effects of alpha- and beta-adrenergic agonists on Trypanosoma cruzi interaction with host cells

We studied the effects of adrenergic agonists on the capacity of blood trypomastigote forms of Trypanosoma cruzi to associate with (i.e., bind and/or penetrate) host cells in vitro. The extent of T. cruzi association with mouse macrophages in the presence of the beta-adrenergic agonist L-isoproterenol was significantly decreased with respect to mock-treated controls. Similar results were obtained when the parasite was pretreated with L-isoproterenol and was then allowed to interact with untreated macrophages. In contrast, pretreatment of trypomastigotes with either L-phenylephrine or methoxamine-alpha-adrenergic agonists--enhanced their reactivity with macrophages. Interaction with a nonphagocytic host cell was also decreased and increased by parasite pretreatment with beta- and alpha-adrenergic agonists, respectively. The L-isoproterenol and L-phenylephrine effects were no longer detectable 2 and 3 hr after their removal, respectively, and were therefore reversible. Atenolol, a specific beta 1 adrenoreceptor blocker inhibited the L-isoproterenol effect, whereas butoxamine, a specific beta 2 blocker, did not. Thus, beta 1-like but not beta 2-like binding sites appeared to be expressed on T. cruzi. Both prazosin and yohimbine, preferential alpha 1- and alpha 2-receptor blockers, respectively, abolished the L-phenylephrine effect. The opposite effects of alpha- and beta-adrenergic agonists suggested that the infectivity of T. cruzi may be regulated by activation of surface components comparable to the adreno-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-interferon inhibits cell infection by Trypanosoma cruzi

Preparations containing alpha/beta-interferon produced by L-929 cells were found to inhibit the capacity of bloodstream forms of Trypanosoma cruzi to associate with and infect mouse peritoneal macrophages or rat heart myoblasts. Marked reductions in the number of parasites per cell as well as in the percentage of cells associated with the trypanosomes were systematically observed in cultures of these cells that contained interferon. The inhibitory effect was abrogated in the presence of specific antibodies against alpha/beta-interferon, and purified beta-interferon induced a similar inhibitory effect, indicating that the active principle in the preparation was indeed interferon. Pretreatment of the parasites with alpha/beta-interferon reduced their infectivity for untreated host cells, whereas pretreatment of either type of host cell had no consequence on the interaction. The effect of interferon on the trypanosomes was reversible; the extent of the inhibitory effect was significantly reduced after 20 min, and was undetectable after 60 min when macrophages were used as host cells. Longer periods of time were required for the inhibitory effect to begin to subside (60 min) and to become undetectable or insignificant (120 min) when rat heart myoblasts were used. The results of additional studies performed with purified preparations of alpha- or beta-interferon revealed that only the latter was inhibitory of cell-parasite association. Because interferon is known to be produced shortly after T. cruzi infection and its administration has been shown to have a marked protective effect against this infection, our results suggest that the latter may involve inhibition of cell infection by interferon.     

The Attraction of Trypanosoma cruzi to Vertebrate Cells In Vitro

SYNOPSIS. A video technic is described that permits a quantification of the degree of attraction of Trypanosoma cruzi trypomastigotes to vertebrate cells in vitro. Bovine embryo skeletal muscle cells (BESM), HeLa cells and Vero cells all attract a myotropic strain of T. cruzi trypomastigotes. BESM cells, however, are 2-fold more attractive to trypomastigotes than HeLa cells and 10-fold more attractive than Vero cells. Heat-inactivation of BESM cells abolishes their ability to respire and also to attract T. cruzi trypomastigotes. As there is no difference in the endogenous oxygen consumption between BESM, HeLa, and Vero cells, it is unlikely that differences in the attraction of trypomastigotes to the 3 cell types are due to variations in the magnitude of pO₂ or pCO₂ gradients in the milieu around the cells.     

Active entry of bloodstream forms of Trypanosoma cruzi into macrophages.

The uptake of bloodstream forms of Trypanosoma cruzi, Y and CL stocks, by mouse peritoneal macrophages and their intracellular differentiation and multiplication has been compared in vitro. After 48 h the number of macrophages showing intracellular amastigote forms was higher when the Y stock was used. The number of parasitized cells increased with the time of contact between parasites and macrophages. Prior treatment of the parasites with anti-T. cruzi antibodies and/or complement increased the number of infected macrophages, but did not interfere with their subsequent differentiation within the macrophages. The number of parasitized cells was greater when macrophages were obtained from mice previously treated with lipopolysaccharide, peptone or thioglycollate. Uptake was not appreciably affected when macrophages were pre-treated with trypsin or anti-macrophage serum, or when the parasites and macrophages were incubated in the presence of cytochalasin B. In the same experimental conditions, epimastigotes of T. cruzi when not able to differentiate into amastigotes. Their uptake was potentiated by previous treatment with specific antibodies and/or complement and was blocked by cytochalasin B. These results confirm that epimastigotes derived from T. cruzi cultures are phagocytosed and suggest that bloodstream forms penetrate actively into macrophages.     

Separation of amastigotes and trypomastigotes of Trypanosoma cruzi from cultured cells

A method is described for the isolation and purification of trypomastigotes and amastigotes of Trypanosoma cruzi from cell cultures. L-A9, a transformed fibroblast cell line, and J774G8, a macrophage-like cell line of tumor origin, were used. Both cell lines were infected with bloodstream trypomastigotes of T. cruzi, which once within host cells transform into dividing amastigotes. After 6--8 days infection the host cells ruptured, spontaneously liberating parasites into the culture medium. L-A9 cells liberated mainly trypomastigotes while J774G8 cells liberated amastigotes. The parasites were collected and purified by centrifugation in a gradient of metrizamide. The purity of the preparation as well as the morphology of the parasites and the host cells were analysed by electron microscopy.     

Substrate specificity of the Trypanosoma cruzi trans-sialidase.

Trypanosoma cruzi trypomastigotes acquire sialic acid (SA) from host glycoconjugates by means of a plasma membrane-associated trans-sialidase (TS). Here we study the substrate specificity of TS, which differs from all known sialyltransferases in that it does not require cytidine monophosphate (CMP)-SA as donor. The T. cruzi TS reversibly transfers SA to saccharides with terminal beta-Gal (but not alpha-Gal) residues. Donors are saccharides with SA linked to terminal beta-Gal residues by (alpha 2-3), but not (alpha 2-6) bonds. The type of beta-linkage of the terminal Gal residue is of minor importance (beta 1-4 and beta 1-6 are slightly better than beta 1-3), whereas chain length and the structure of additional vicinal sugar residues are not relevant. SA on the surface of living trypomastigotes of T. cruzi is transferred back and forth between the parasite surface and acceptor molecules with terminal beta-Gal, either in solution or on the surface of neighbouring mammalian cells. Addition of fucose residue on or close to the terminal galactose impairs TS activity. As a consequence, the enzyme acts poorly on the E-selectin ligand sialyl-Lewisx and its precursor Lewisx, and in vitro adhesion of TS-treated neutrophils to L-cells expressing L-selectin is not affected. Modifications in the structure of the (alpha 2-3)-linked N-acetyl-neuraminic acid (Neu5Ac) (deoxy or methoxy) of the donor molecules do not impair transfer if the changes are at C9, whereas changes at C4, C7 and C8 impair the ability to donate the modified SA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trypanosoma cruzi: methoprene is a potent agent to sterilize blood infected with trypomastigotes

The effects of methoprene, a juvenile hormone analogue (JHA), on Trypanosoma cruzi bloodstream trypomastigotes (Tulahuen strain, Tul 2 stock) were studied. It was observed that 150microM of methoprene in in vitro experiments cause cellular death of T. cruzi.In contrast, methoprene was not able to clear bloodstream trypomastigotes in in vivo experiments, but it was observed a decrease of parasitemia levels of infected mice treated with 200microg of methoprene/mouse/day during 5 days. According to these results and the low toxicity of methoprene, we suggest that this compound will serve as an effective agent to sterilize blood for transfusions.     

A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells.

When trypomastigotes of T. cruzi emerge from cells of the mammalian host, they contain little or no sialic acids on their surfaces. However, rapidly upon entering the circulation, they express a unique cell surface trans-sialidase activity. This enzyme specifically transfers alpha (2-3)-linked sialic acid from extrinsic host-derived macromolecules to parasite surface molecules, leading to the assembly of Ssp-3, a trypomastigote-specific epitope. The T. cruzi trans-sialidase does not utilize cytidine 5' monophospho-N-acetylneuraminic acid as a donor substrate, but readily transfers sialic acid from exogenously supplied alpha (2-3)-sialyllactose. Monoclonal antibodies that recognize sialic acid residues of Ssp-3 inhibit attachment of trypomastigotes to host cells, suggesting that the unusual trans-sialidase provides Ssp-3 with structural features required for target cell recognition.     

Failure of Trypanosoma cruzi to trigger the respiratory burst of activated macrophages. Mechanism for immune evasion and importance of oxygen-independent killing

Activated macrophages are thought to kill Trypanosoma cruzi, which lack catalase, by the generation of hydrogen peroxide. We investigated triggering of the respiratory burst of activated macrophages induced by phagocytosis of virulent T. cruzi, bloodform trypomastigotes, amastigotes obtained from spleens, and tissue culture organisms; and of relatively nonvirulent epimastigotes. All stages of T. cruzi prompted the release of less than 10% of hydrogen peroxide released by activated macrophages when stimulated with PMA or Candida. Superoxide anion production was not stimulated by PMA or Candida in activated macrophages nor was there a significant qualitative reduction of nitroblue tetrazolium induced by ingestion of virulent T. cruzi. Opsonization of T. cruzi with specific antibody did not promote the release of hydrogen peroxide or the reduction of nitroblue tetrazolium. Similar results were observed with activated spleen macrophages. Incubation of activated macrophages with catalase, catalase and superoxide dismutase, sodium benzoate with or without catalase, and respiratory burst-exhausting PMA failed to inhibit the killing of T. cruzi in vitro. These results indicate that 1) virulent opsonized or unopsonized T. cruzi do not trigger a respiratory burst by activated macrophages and 2) oxygen-independent killing of T. cruzi is of prime importance.     

Interaction of Trypanosoma cruzi with macrophages: effect of previous incubation of the parasites or the host cells with lectins

The effect of incubation with lectins of the macrophages or two evolutive stages of Trypanosoma cruzi (noninfective epimastigotes and infective trypomastigotes) on the ingestion of the parasites by mouse peritoneal macrophages was studied. Lectins which bind to residues of mannose (Lens culinaris, LCA), N-acetyl-D-glucosamine or N-acetylneuraminic acid (Triticum vulgaris, WGA), beta-D-galactose (Ricinus communis, RCA), N-acetyl-D-galactosamine (Phaseolus vulgaris, PHA; Dolichos biflorus, DBA; and Wistaria floribunda, WFA), fucose (Lotus tetragonolobus, LTA), and N-acetylneuraminic acid (Limulus polyphemus, LPA) were used. By lectin blockage we concluded that, alpha-D-mannose-like, beta-D-galactose and N-acetyl-D-galactosamine (PHA, reagent) residues, located on the macrophage's surface are required for both epi- and trypomastigote uptake, while N-acetylneuraminic acid and fucose residues, impede trypomastigote ingestion but do not interfere with epimastigote interiorization. Macrophages' N-acetyl-D-glucosamine residues are required for epimastigote uptake. On the other hand, from the T. cruzi surface, mannose residues prevent ingestion of epi- and trypomastigotes. Galactose residues participate in endocytosis of trypomastigotes, but hinder epimastigote interiorization. Exposed N-acetyl-D-glucosamine residues are required for uptake of the two evolutive forms. N-acetylneuraminic acid residues on the trypomastigote membrane prevent their endocytosis by macrophages. These results together with those reported previously showing the effect of monosaccharides on the T. cruzi-macrophage interaction, indicate that (a) sugar residues located on the parasite and on macrophage surface play some role in the process of recognition of T. cruzi, (b) different macrophage carbohydrate-containing receptors are involved in the recognition of epimastigotes and trypomastigotes forms of T. cruzi, (c) N-acetylneuraminic acid residues located on the surface of trypomastigotes or macrophages impede the interaction of the parasite with these host cells, and suggest that (d) sugar-binding proteins located on the macrophage surface participate in the recognition of beta-D-galactose and N-acetyl-D-galactosamine residues located on the surface of trypomastigotes and exposed after blockage or splitting off of N-acetylneuraminic acid residues. Some lectins which bind to macrophages and block the ingestion of parasites did not interfere with their adhesion.     

The activity of nifluridide on reduviids and rodent and human trypanosomes

The ability of nifluridide to kill reduviids was assayed in mice fed 7 ppm in diet and on cattle injected subcutaneously at 5 mg/kg body weight. Nifluridide was systemically active against Triatoma infestans on mice and Rhodnius prolixus on cattle. No effects on Trypanosoma (Schizotrypanum) cruzi could be detected in the intestinal contents of Triatoma infestans killed by the compound. In vitro and in vivo studies were conducted to determine the effects of nifluridide on trypanosomes growing in medium and in experimentally infected mice. Culture forms of Trypanosoma cruzi grown at 27 degrees C that are morphologically similar to epimastigotes found in infected bugs were affected by 2.5 to 10 ppm in the medium. Mice fed nifluridide in the diet simultaneous with infection of Trypanosoma cruzi or Trypanosoma (Herpetosoma) musculi exhibited parasitemias and tissue infections similar to nontreated infected mice. At the concentration tested, bloodstream trypomastigotes and culture epimastigotes of Trypanosoma musculi were unaffected by nifluridide. Only the culture epimastigotes of Trypanosoma cruzi were affected by the drug but not the bloodstream and tissue forms.